Insulin resistance, hepatic inflammation, and NAFLD are interlocking pathophysiologic events, but the mechanisms of these abnormalities, and the ways in which these different processes interact, are poorly understood. This is a broad, collaborative application in which the four participating PI's and laboratories will concentrate their focus on the etiology and pathophysiology of hepatic inflammation, steatosis, and insulin resistance. The scale of this application is substantial and will focus on four overall specific aims. In the first aim, an ambitious, large scale time course will be undertaken in high fat diet (HFD)/obese mice, coupled with systematic in vitro and in vivo measurements to uncover the dynamic temporal time course and key transition points enabling the development of hepatic insulin resistance/inflammation/steatosis. The second aim explores a novel hypotheses which proposes that changes in intestinal microflora and gut permeability to bacterial products triggers inflammatory signals directed to the liver. These inflammatory stimuli then interact with immune cells in the liver, generating the chronic hepatic inflammatory state. The third aim encompasses several new ideas and hypotheses aimed at delineating the molecular mechanisms underlying the metabolic disturbances in the liver. These studies will involve tracking the itinerary of immune cells to the liver, identifying the phenotypic function of the different liver cell types, studies of biochemical pathways involved in insulin signaling, lipogenesis/fat oxidation, inflammation, and the identification of transcription factor cistromes and epigenetic changes in genomic loci induced by obesity. In vivo and in vitro studies in a number of knockout mice will be heavily used in the pursuit of these studies. The final aim proposes translational studies in which liver biopsies will be obtained from obese NAFLD subjects before and after weight loss. Cellular, biochemical, and genomic studies will be performed in these biopsies and correlated with the in vivo clinical data on these patients. In this way, we will be able to test the ideas and concepts learned from the basic studies in the first three aims for relevance to human pathophysiology. PUBLIC HEALTH RELEVANCE: NAFLD is closely associated with hepatic insulin resistance and inflammation and is the most common liver disease in the US. The pathophysiologic mechanisms underlying the interactions between hepatic insulin resistance, inflammation, and steatosis are poorly understood, and this project should lead to a greatly improved basic understanding of this disorder with the potential to lead to new therapeutic opportunities.